For topical treatment of skin cancer, the design of pH-responsive nanocarriers able to selectively release the drug in the tumor acidic microenvironment represents a reliable option for targeted delivery. In this context, a series of newly synthesized surface-active fatty acid-protic ionic liquids (FA-PILs), based on tetramethylguanidinium cation and different natural hydrophobic fatty acid carboxylates, have been investigated with the aim of developing a pH-sensitive nanostructured drug delivery system for cutaneous administration in the skin cancer therapy. The capability of FA-PILs to arrange in micelles when combined with each other and with the non-ionic surfactant d-α-Tocopherol polyethylene glycol succinate (vitamin E TPGS) as well as their ability to solubilize imiquimod, an immuno-stimulant drug used for the treatment of skin cancerous lesions, have been demonstrated. The FA-PILs-TPGS mixed micelles showed pH-sensitivity, suggesting that the acidic environment of cancer cells can trigger nanostructures’ swelling and collapse with consequent rapid release of imiquimod and drug cytotoxic potential enhancement. The in vitro permeation/penetration study showed that the micellar formulation produced effective imiquimod concentrations into the skin exposed to acid environment, representing a potential efficacious and selective drug delivery system able to trigger the drug release in the tumor tissues, at lower and less irritating drug concentrations. The incidence of skin cancer is on the rise worldwide and surgical removal or invasive procedures represent the most widely used treatments. In recent years, numerous therapeutic strategies have been introduced, such as immunotherapy, targeted therapy, photodynamic therapy, and gene therapy ,2,3,4. WThe new strategies for drug development include the use of existing drugs in the formulation of sustainable and biodegradable nanosystems for specific target delivery. This approach has the potential for reducing time and costs of developing new drugs for clinical use. However, the controlled release of the drug from the nanostructures represents a key point in the architecture of new drug delivery systems, taking into account that drug release can be influenced by many factors ,19. In oIonic liquids (ILs) are organic salts with melting points below 100 °C which are endowed with unique physico-chemical properties such as low flammability, negligible vapor pressure, high thermal and chemical stability, and wide electrochemical window ,26,27,28nCO-HTMG; (nCO) to behave as surfactants and form micelles with dimensions in the nanometer range. The ultimate aim was to develop a pH-sensitive nanostructured drug delivery system for cutaneous administration of drugs against skin cancer. Moreover, the effect of FA-PILs on the physico-chemical properties of a non-ionic aqueous surfactant, such as d-α-Tocopherol polyethylene glycol succinate (vitamin E TPGS), has been evaluated. Indeed, it has been reported that surfactants are able to form various molecular aggregates in ILs  quinolin-4-amine .The following materials were used: d-α-Tocopherol polyethylene glycol succinate .All other reagents were analytical grade. Ultra-pure water was prepared using Milli-QTetradecanoic acid (98%) was obtained from Alfa Aesar. , 1,1,3,3-tetramethylguanidine (99%), dodecanoic acid (≥99%), decanoic acid (≥99.5%), and octanoic acid (≥99%) were purchased from Sigma Aldrich .1H NMR.An equimolar amount of fatty acid was added to 1,1,3,3-tetramethylguanidine at room temperature, without any solvent addition. The reaction mixture was heated to 60 °C and stirred for 1 h. At the end of the reaction, the mixture was cooled to room temperature to obtain a white solid product. The obtained product was analyzed by 7CO-HTMG. The preparation of C7CO-HTMG  was performed according to the general procedure. 1H NMR (CDCl3) 8.80 , 2.89 , 2.07 , 1.55–1.45 , 1.25–1.09 , 0.78 .Tetramethylguanidinium octanoate C9CO-HTMG. The preparation of C9CO-HTMG  was performed according to the general procedure. 1H NMR (CDCl3) 8.35 , 2.95 , 2.16 , 1.65–1.49 , 1.34–1.16 , 0.86 .Tetramethylguanidinium decanoate C11CO-HTMG. The preparation of C11CO-HTMG  was performed according to the general procedure. 1H NMR (CDCl3) 8.83 , 2.89 , 2.09 , 1.58–1.44 , 1.28–1.09 , 0.81 .Tetramethylguanidinium dodecanoate C13CO-HTMG. The preparation of C13CO-HTMG  was performed according to the general procedure. 1H NMR (CDCl3) 8.13 , 2.94 , 2.15 , 1.55–1.48 , 1.36–1.11 , 0.86 .Tetramethylguanidinium tetradecanoate C1H and 13C NMR spectra were recorded with a Bruker Avance II operating at 400 MHz at 24 °C. For the first-order proton chemical shifts CDCl3 , the chemical shifts are given in δ. The following abbreviations are used: s = singlet, m = multiplet, bs = broad singlet, t = triplet, bt = broad triplet, q = quartet, qui = quintuplet, sext = sextet.h = 2–20 °C/min). DSC experiments were carried out in duplicate.The thermal behavior of the ionic liquids was analyzed by a differential scanning calorimeter  following the procedure reported in Mezzetta et al. [Surface tension measurements were carried out by the use of a Krüss K12 tensiometer . The surface tension was determined with the single-measurement method using deionized water for instrument calibration (surface tension: 72.8 mN/m at 25.0 ± 0.1 °C). The platinum plate used in the measurements was cleaned with ultra-pure water (Millipore) followed by heating through flame. The surface tension of each solution was measured by successive addition of different aliquots of a stock solution to ultra-pure water after careful mixing and equilibration. All measurements were repeated at least two times.7CO-HTMG, C9CO-HTMG, C9CO-HTMG, and C13CO-HTMG, respectively) were prepared and subsequently mixed in order to maintain a fixed amount of FA-PIL with varying that of vitamin E TPGS.In the case of FA-PILs/TPGS binary systems, stock solutions of the surfactants at the same molar concentration  and the surface tension at the cmc (γcmc) were determined from the intersection of the two straight lines obtained by surface tension curves in low- and high-concentration regions (γ-log C curves) using a linear regression analysis method.The values of the critical micellar concentration  were prepared by introducing the selected FA-PIL  in water, stirring overnight.Self-assembling micelles containing FA-PILs (CnPILs-TPGS/Nano and CnPILs-TPGS-IMQ/Nano), the selected FA-PIL and vitamin E TPGS were heated over the melting point of both surfactants (50 °C); then, drug was added under continuous stirring to obtain a homogeneous blend. As a last step, water at the same temperature as the blend was added and the mixture was stirred overnight.In the case of mixed self-assembling micelles  to remove unloaded drug, aggregates, and foreign particulates.In all cases, the final mixture containing IMQ was filtered through cellulose acetate filters  following the procedure reported in Terreni et al. [The prepared micelles were characterized with respect to size, size distribution, and encapsulation efficiency (drug loading and entrapment). Size and size distribution of the micelles were determined by using a Dynamic Light Scattering (DLS) Beckman Coulteri et al. .The average size for each formulation was determined on 6 runs of three different samples at two angles (62.6° and 90°) and run time of 200 s at 20 °C.The entrapment efficiency, defined as the percentage of drug loaded inside the micelles with respect to the one formerly added to the formulation, was determined by high performance liquid chromatography (HPLC). To ensure complete destruction of the micelles and release of the incorporated drug, an aliquot of each formulation was diluted with methanol and the amount of drug was determined by HPLC analysis.The entrapment and loading efficiency of IMQ were calculated using the following equations:The pH sensitivity of the nanostructures was investigated by testing the distribution of the particle size in both the simulated endo/lysosome conditions (pH = 5.5) and physiological pH (pH = 7.4) at fixed time intervals .In order to simulate the tumoral and endosomal environment, the in vitro release profiles of IMQ from the prepared micelles has been examined at pH 7.4  and pH 5.5  using a dynamic dialysis method.Briefly, the obtained micellar systems were dispersed in 1 mL of different buffer solution pH 7.4  and pH 5.5 (sodium acetate buffer) and placed in a dialysis bag . The receptor medium consisted of 5 mL of buffer at the same pH maintained at 37 °C (body temperature) in a water bath with gentle stirring (20 rpm). At predetermined time intervals, 1 mL aliquots were withdrawn and replaced with an equal volume of fresh medium. All the experiments lasted 24 h and were performed in triplicate. The amount of drug in the samples was determined by HPLC.7/C13CO-HTMG-TPGS/Nano were compared. 501Mel cells were kindly provided by Dr. Poliseno .The cytotoxicity on 501Mel melanoma cells of IMQ alone and that of IMQ loaded on non-cytotoxic concentrations of C7/C13CO-HTMG-TPGS-IMQ/Nano alone and loaded with IMQ were dissolved in water.IMQ was dissolved in DMSO and the C7/C13CO-HTMG-TPGS-IMQ/Nano at two different concentrations for 24 h. At the end of the treatment, 20 μL of MTS solution was added to each well and absorbance was read at 490 nm using the Infinite M200 NanoQuant instrument . Optical density values from vehicle-treated cells were considered as 100% cell viability. For each treatment, the corresponding vehicle-treated cells were used as control, i.e., the treatment with DMSO alone at the corresponding concentration of IMQ represents the control of IMQ and cell medium represents the control of C7/C13CO-HTMG-TPGS-IMQ/Nano.Culture conditions and cell viability assay -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTS) were performed in agreement with the ones reported in Carpi et al. . After o® commercial product , Italy), cream containing 5% IMQ, used as a reference. The receiving phase consisted of either PBS pH 7.4 added with 1% serum albumin to enhance drug solubility or sodium acetate buffer pH 5.5 , maintained at 37 °C and stirred at 600 rpm. At predetermined time intervals, the receiving phase (5 mL) was withdrawn for HPLC analysis and replaced with the same volume of fresh fluid, and sink conditions were maintained throughout the entire study. All experiments lasted 24 h and were replicated four times.Porcine ears skin, used as a model, was obtained from freshly sacrificed animals in a local slaughterhouse as previously described in Tampucci et al. . The pigAt the end of the permeation experiments, the skin was collected and treated following the procedure reported and validated in Monti et al. . BrieflyIMQ-Loaded FA-PILs-TPGS mixed nanostructures were subjected to the freeze-drying process and the freeze-dried samples were stored in a desiccator for up to 30 days. The freeze-drying process was carried out in a Virtis apparatus  as previously reported in Burgalassi et al. .The freeze-dried samples, reconstituted with the original volume of water to restore the initial formulation, were subjected over time (15 and 30 days) to dimensional and HPLC analysis, to highlight, respectively, any changes in particle size and in drug loading with respect to the same formulation before freeze-drying. The experiments were performed in triplicate.®, Italy) was used. Isocratic elution was performed using a mobile phase consisting of a mixture of CH3OH/CH3CN/H2O/TEA (180/270/530/20) filtered through a 0.45 μm pore size membrane filter. The detection wavelength was 242 nm, the flux was 0.5 mL/min, and the retention time was 6 min.A reverse-phase-HPLC method was used to determine the IMQ concentration in the samples with the apparatus previously described in Tampucci et al. . The injThe amount of IMQ in the samples was determined by comparison with appropriate external standard curves obtained applying the least square linear regression analysis. For in vitro studies, the calibration curves were obtained by dissolving the drug in methanol and then diluting with the appropriate medium (buffer solutions at pH 5.5 or 7.4). In case of skin samples, the standard curves were obtained by adding increasing amounts of the drug to a blank biological matrix, as previously described .p-value < 0.05 was considered statistically significant.Data related to in vitro cytotoxicity analysis were reported as mean ± standard deviation (SD) of at least three independent experiments, each performed in triplicate. Statistical analysis was performed using GraphPad Prism software, version 8.0  using the t-test. A L), as the time needed by the drug to saturate the membrane and to reach the receiving phase, estimated from the X-intercept of the regression line, and drug cumulative amount permeated at end of the 24 h (Q24h). Furthermore, the drug content (Qskin) retained in the skin at the end of the experiment was calculated following the extraction procedure. A retention factor (RF), calculated as Qskin/Cv, is reported to compare the amount of drug retained in the skin after application of each formulation, taking into account the initial concentration of the drug (Cv).For the in vitro permeation/penetration study, linear regression analysis of pseudo steady-state diffusion plots allowed calculation of the following parameters: steady-state flux (J), as Q/At, in which Q is the amount of drug diffusing across the area A in the time t; lag time (tp < 0.05).The significance of the differences between permeation parameters was assessed by GraphPad Prism software. Group comparison was assessed by using the Student’s two-tailed unpaired t-test  and of the methylene group adjacent to the carboxylate group (2.07–2.16 ppm) confirmed the 1:1 ratio between cation and anion.The FA-PILs structures were assessed by analyses . The hig7CO-HTMG, which features the shortest alkyl chain of the series, exhibits a type I thermal behavior at 2 °C/min that shifts towards the type III behavior at higher scanning rates (5 and 10 °C/min), without the presence of polymorphic events  at different scanning rates  in the temperature range going from −90 to 100 °C under a nitrogen flow. Three general thermal behaviors have been found for ILs and are classified as: type I behavior for ILs which have little tendency to crystallize and show only glass transitions, type II for those ILs which form crystals on cooling and display the related melting event during the heating, thus acting as low melting salts, and type III when cold crystallization and melting are both recorded during the heating half cycle ,71. For c events a. C9CO-Hing ramp b–d, withThe surface tension of FA-PILs was measured to evaluate their surface activity. cmc1) represented by a plateau region in the surface tension isotherms, followed by a second plateau (γcmc2) indicating a second cmc value. The values of cmc1 and cmc2 for each FA-PILs are shown in cmcs, which decrease as the alkyl chain increases, can be observed, confirming previously reported data for similar structures [It is apparent that the surface tension of each FA-PIL gradually decreases by increasing the IL concentration, first, to reach a nearly constant value , aggregation of the micelles in more complex structures may occur. Each added surfactant molecule aggregates into small micelles, until the second cmc (cmc2) is reached; after that, the further added amphiphiles begin to organize themselves into larger non-surface-active linear micelles [Generally, the micelles have a spherical shape at surfactant concentrations higher than micelles ,75.Finally, it is important to highlight the absence of a minimum in the breakpoint region that generally occurs in presence of highly surface-active impurities. This is further pointed out by sharp endothermic peaks obtained in the DSC experiments, which confirms the purity of the synthesized FA-PILs ,77.cmc of vitamin E TPGS (0.23 mg/mL) was found in good agreement with the literature value for the pure amphiphile (0.02% w/w).Mixing of surfactants with ILs endowed with surfactant properties can considerably alter the physico-chemical properties of the system . In the cmc values for vitamin E TPGS in the binary mixtures (C7CO-HTMG/TPGS and C9CO-HTMG/TPGS) were higher than that of the vitamin alone . Conversely, when FA-PILs based on longer chain fatty acids, such as lauric and myristic acids (C11CO-HTMG and C13CO-HTMG), were employed in the binary mixtures, the cmc of vitamin E TPGS was lower than that of the single component (0.043 and 0.014 mg/mL), probably due to an increase in hydrophobic interactions. Indeed, vitamin E TPGS-, the C12-, and the C14 fatty acids-based ionic liquids all possess a distinct hydrophobic character, which can favor the micellization process.It is known that micelles formation is governed both by electrostatic interactions of the polar head of the surfactants and by the hydrophobic interactions of the hydrocarbon chains . When thcmc1. A pool of formulations was tuned up and the composition of the mixtures along with their physico-chemical characterization in terms of hydrodynamic diameter (Dh) at two different angles (62.6° and 90°) and polydispersity index (P.I.) is reported in All the synthesized FA-PILs were screened for their capability to originate micelles when dispersed in water at a concentration above their 9CO-HTMG, C11CO-HTMG, and C13CO-HTMG, micelles of dimensions around 200 nm and with a uniform size distribution at the two angles tested were obtained only when the concentration of ionic liquid was between cmc1 and cmc2. Conversely, in the case of C7CO-HTMG, a population of nano-sized micelles was formed only above the cmc2, probably due to the IL’s shorter alkyl chain. In any case, the dimensional distribution obtained with the formulation C7CO-HTMG/Nano showed a high polydispersity index (P.I. = 0.709) indicative of a not homogeneous size. Furthermore, it was impossible to detect the formation of a colloidal structure at the 62.6° angle.In the case of Ch in the range from 7.67 to 42.37 nm) with respect to the system containing only the FA-PIL. In particular, it is interesting to highlight that it was possible to use the FA-PIL under its cmc2 by mixing C7CO-HTMG and vitamin E TPGS (C7CO-HTMG-TPGS/Nano). By adopting this strategy, micelles with a low Dh and a narrow dimensional distribution at the two angles tested could be obtained, which is indicative of a spherical shape of the colloidal system. Conversely, when C11CO-HTMG was used in the binary system, a not homogeneous dispersion was obtained (P.I. between 1.163 and 1.322), probably due to both the FA-PIL concentration near the cmc2 and the high molar ratio between the two ionic and non-ionic surfactants (1:10 molar ratio).On the basis of the results obtained with both the preliminary formulation study and the surface tension analysis, mixed systems containing FA-PILs and vitamin E TPGS were prepared with the aim of decreasing the dimension of the micelles and exploiting the capacity of vitamin E TPGS to inhibit the MDR. The prepared formulations are reported in 7CO-HTMG and C13CO-HTMG with vitamin E TPGS. C7/C13CO-HTMG-TPGS/Nano showed the best characteristics in terms of both Dh and P.I. . Regarding the structure of the above-mentioned micelles, a spherical shape could be hypothesized (10.1 and 10.3 nm at 62.6° and 90° angles). Besides, as HTMG presents a positive charge and a triangular shape, it can act as a hydrogen bond fitting using acidic hydrogen on NH2 and CH3 groups. A reasonable hydrogen-bonded cluster is represented in Finally, a ternary system designed with our best results in mind has been formulated by mixing C7/C13CO-HTMG-TPGS/Nano have been selected as a delivery system of IMQ. The topical delivery of IMQ is extremely challenging due to its very low aqueous solubility in water medium (0.01 mM) and poor skin permeation capacity, despite its low molecular weight (MW 240.30 g/mol) and a logP of 2.6 [CP of 2.6 . Therefo13CO-HTMG, alone or in combination with vitamin E TPGS , the obtained micelles presented a Dh in the range 206–269 nm ; on the other hand, the hydrodynamic diameter of the C13CO-HTMG-TPGS-IMQ/Nano markedly increased due to IMQ encapsulation (269.5 and 218.7 nm vs. 15.5 and 14.0 nm for loaded and empty micelles).It should be noticed that there were no differences in D7C13CO-HTMG-TPGS-IMQ/Nano), only a slight increase in both the Dh and P.I. was noticed with respect to the empty micelles, maybe because of the lower entrapment efficiency (EE%) of C7C13CO-HTMG-TPGS-IMQ/Nano with respect to C13CO-HTMG-TPGS-IMQ/Nano.Conversely, when the ternary system containing IMQ was formulated  with respect to the value reported in the literature [Regardless, it is worth highlighting that in all cases, notwithstanding the low EE%, the drug solubility was enhanced from 3.6-fold to 83-fold  is employed as part of the FA-PIL, possibly on account of the weaker hydrophobic interactions, which in turn facilitate the collapse of the micelle.The release of IMQ from Cmulation . The des7C13CO-HTMG-TPGS-IMQ/Nano was further investigated to demonstrate the structural changes of these hybrid micelles in an acidic environment. The concerned micelles were incubated in a pH 5.5 buffer solution simulating the tumor acidic/endosomal environment and the Dh and size distribution were determined at pre-determined time intervals, comparing to those measured at physiological pH. At the zero time point, the micelles were in a narrow size range with low P.I.; subsequently, after the exposure to acidic conditions, both the particles’ size and the percentage of larger particles gradually increased  showed that they did not induce a significant effect on cells’ viability after 24 h of incubation up to a concentration limit of each component related to their amount in the formulation: C7CO-HTMG 86.6 g/mL, C13CO-HTMG 3.33 g/mL, and vitamin E TPGS 504 g/mL. The C7/C13CO-HTMG-TPGS-IMQ/Nano were tested in the same conditions and the IMQ concentration in the well was 200 ng/mL. The effect of this concentration and its 1/10 were compared to the one of the IMQ alone.Imiquimod is an anticancer agent showing both immune-stimulation activity and a direct cytotoxic/anti-proliferative action ,81,82,83As shown in 7C13CO-HTMG-TPGS-IMQ/Nano was further investigated in a set of in vitro permeation studies using pig ear skin as a model. These experiments were carried out to evaluate the ability of the micellar formulation to deliver the drug selectively into the skin, where the target site for the topical treatment of melanoma is located, while reducing systemic absorption to a minimum.The behavior of the formulation C® was taken as a term of comparison.The results of the in vitro experiments are summarized in ® and C7C13CO-HTMG-TPGS-IMQ/Nano tended to produce a significantly greater permeation at pH 5.5 with respect to physiological pH. Any significant difference in the permeation parameters was found between the two formulations tested at each pH. The pH-sensitive behavior of the self-assembling mixed micelles could be responsible for the higher amount of drug permeated in acidic conditions, as well as the dimensions of the nanostructure having a Dh 20 nm could favor the permeation of IMQ through intact skin, confirming data in Reference [It is possible to observe that both tested formulations produced a transcutaneous permeation of drug dependent on the pH of the receiving phase; in particular, Imunocareeference . On the eference .One of the greatest challenges in topical application of anticancer drugs is to obtain a drug amount in the skin sufficient to give a therapeutical effect. Nanocarriers could improve drug targeting due to their ability of addressing the tumor cells, contemporaneously markedly reducing the amount of drug applied on the skin, and avoiding the direct contact of the drug with the cutaneous surface .7C13CO-HTMG-TPGS-IMQ/Nano produced a significantly higher amount of IMQ retained in the skin at the acidic tumor pH with respect to that obtained at physiological pH, while for the commercial formulation, any difference in the behavior at the two pHs was detected. The commercial formulation produced a 26.8-fold (pH 7.4) and 7.3-fold (pH 5.5) higher amount of drug retained in the skin with respect to C7C13CO-HTMG-TPGS-IMQ/Nano. However, it is worth mentioning that when the micellar formulation was applied on the skin, the amount of IMQ administrated was about 1300-fold lower with respect to Imunocare® , suggesting that the nanostructured formulation was able to improve drug partitioning through the skin, as evident from the histograms in An important issue for a possible application on tumor tissue is that the micellar formulation C7C13CO-HTMG-TPGS-IMQ/Nano is a colloidal dispersion in liquid form, which in the future will need to be formulated with a rheological agent in order to be applied on the skin surface favoring an intimate contact of the drug with the skin. Anyway, the amount of IMQ (5.93 ± 0.64 g/mL) retained in the skin at pH 5.5 with the micellar formulation was definitely higher than both the 200 ng/mL found to be effective in the in vitro cell viability assay performed on 501Mel melanoma cells (paragraph 3.6) and the IC50 value reported in literature (2 M) [Besides, it should be noted that the commercial formulation is a semisolid formulation, while Cre (2 M) . Moreove7C13CO-HTMG-TPGS-IMQ/Nano formulation was subjected to the lyophilization process in order to stabilize the nanostructured system during storage for subsequent redispersion in water at the time of use. The freeze-dried sample obtained showed a soft appearance and was readily redispersible with deionized water to form a clear and colorless solution. The results of the dimensional analysis on reconstituted lyophilizates are shown in The freeze-drying process has been extensively investigated to stabilize a variety of nanocarriers, but only a few reports deal with lyophilization of micellar systems . The C7CThe design of new biodegradable, nontoxic, and pH-sensitive polymeric materials, suitable to be used as drug delivery systems, is a great challenge for the pharmaceutical community. Moreover, one of the objectives in the above-mentioned field is to improve the effectiveness in tumor targeting, by selectively addressing cancer tissues and sparing healthy ones. The combination of newly synthesized fatty acid-protic ionic liquids (FA-PILs) and vitamin E TPGS led to the formation of self-assembling micelles of nanometric size, able to encapsulate the anticancer drug IMQ and to release it in cancer tissues with a pH-triggered mechanism; besides, the proposed nanosystem is capable of producing effective drug amounts at the action site  following topical application, thus representing a potential drug delivery system for skin cancer treatment.


The purpose of the current study was to evaluate the analytical performance of seven kits for detecting IgM/IgG antibodies against coronavirus (SARS-CoV-2) by using four chemiluminescence immunoassay systems.Fifty patients diagnosed with SARS-CoV-2 infection and 130 controls without coronavirus infection from the General Hospital of Chongqing were enrolled in the current retrospective study. Four chemiluminescence immunoassay systems, including seven IgM/IgG antibody detection kits for SARS-CoV-2 , were employed to detect antibody concentrations. The chi-square test, the receiver operating characteristic (ROC) curve and Youden’s index were determined to verify the cut-off value of each detection system.The repeatability verification results of the A, B, C and D systems are all qualified. D_Ab performed best (92% sensitivity and 99.23% specificity), and B_IgM performed worse than the other systems. Except for the A_IgM and C_IgG systems, the optimal diagnostic thresholds and cut-off values of the other kits and their recommendations are inconsistent with each other. B_IgM had the worst AUC, and C_IgG had the best diagnostic accuracy. More importantly, the B_IgG system had the highest false-positive rate for testing patients with AIDS, tumours and pregnancies. The A_IgM system test showed the highest false-positive rates among elderly individuals over 90 years old. COVID-2019 IgM/IgG antibody test systems exhibit performance differences.The Innodx Biotech Total Antibody serum diagnosis kit is the most reliable detection system for anti-SARS-CoV-2 antibodies, which can be used together with nucleic acid tests as an alternative method for SARS-CoV-2 detecting. SARS-CoV-2 is highly contagious through aerosols, droplets and contact.2 Generally, the incubation period of SARS-CoV-2 is three to seven days, but the longest incubation period can reach 14 days.3 It has caused more than 7,250,000 human infections and nearly 410,000 deaths worldwide as of the end of 9 June. Therefore, the early diagnosis of SARS-CoV-2 infection is crucial. Previous studies have shown that the SARS-CoV-2 antigen stimulates the immune system to produce an immune response and that specific IgM and IgG antibodies appear in the serum of patients after infection.4 The SARS-CoV-2-specific IgM and IgG antibody tests have been involved in the diagnosis criteria for suspected patients whose COVID-19 viral nucleic acid test appears false negative, according to the recently published guidelines of Novel Coronavirus Pneumonia Diagnosis and Treatment , which were advocated by the National Health Committee.5Coronavirus pneumonia  is an acute respiratory infection caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2).6 Chemiluminescence immunoassays are a laboratory technology that combines a luminescence system with an immune response. It not only uses the specificity of the immune response but also has the high sensitivity of the luminescence reaction and is widely used in immunoassays.7 Our laboratory currently has four automatic chemiluminescence immunoassay systems, A, B, C and D, of which the three detection systems A, B and C detect SARS-CoV-2-specific IgM and IgG antibodies, and the D system detects total IgM/IgG antibodies. The current investigation intends to evaluate the repeatability, clinical sensitivity and specificity of seven antibody detection kits for four detection systems, as well as the false-positive rate in special populations. Youden’s index verifies the best diagnostic threshold  of each detection system to understand the analytical detection performance of each system and ensure the detection results.Current popular detection methods for anti-SARS-CoV-2 antibodies include colloidal gold and chemiluminescence immunoassays.9Fifty serum samples from patients with SARS-CoV-2 infection diagnosed in 26 January to 6 February 2020 and 130 serum samples from patients with other conditions, including 20 late-term pregnant women, 20 patients with solid tumours, 20 patients with AIDS, 21 patients over 90 years old and 49 normal controls, were enrolled from the Immunology Department of the Laboratory Department of Chongqing General Hospital  from late February to March 2020. Control populations are selected based on common false-positive populations  reported in the daily work and literature reports. All patients with SARS-CoV-2 infection were confirmed by nucleic acid testing (NAT) and computed tomography (CT) scan. All collected serum specimens were inactivated in a water bath at 56°C for 1 h and then stored in a freezer at –80°C.The automatic immunochemiluminescence analyser A was called detection system A . Reagents included the anti-coronavirus (SARS-CoV-2) IgM antibody detection kit , and S/CO  ≥1.0 was designated positive. For the SARS-CoV-2 IgG antibody detection kit , S/CO ≥ 1.0 was designated positive. The fully automatic immunochemiluminescence analyser B was called detection system B . Reagents included the SARS-CoV-2 IgM antibody detection kit , for which S/CO ≥ 1.0 AU/ml was designated positive. For the SARS-CoV-2 IgG antibody detection kit , S/CO ≥ 1.0 AU/ml was designated positive. The automatic immunochemiluminescence analyser C was called detection system C . Reagents included the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgM antibody detection kit , for which S/CO ≥ 10 AU/ml was designated positive. For the SARS-CoV-2 IgG antibody detection kit , S/CO ≥ 10 AU/ml was designated positive. The fully automatic immunochemiluminescence analyser D was called detection system D . Reagents included the SARS-CoV-2 antibody detection kit , for which S/CO ≥ 1.0 was designated positive.Under the conditions of calibration and quality control of the detection systems, all of them are qualified, and the following experiments are carried out. The cut-off value is 1.0, 1.0, 1.10 AU/ml, 1.10 AU/ml, 10 AU/ml, 10 AU/ml and 1.0 in the A_IgM, A_IgG, B_IgM, B_IgG, C_IgM, C_IgG and D_Ab detection systems, respectively. Among 50 specimens of patients infected with SARS-CoV-2, one case of a weakly positive specimen with an S/CO value less than three times the cut-off value  and one case with an S/CO value greater than three times the cut-off value  were selected. Within-run precision was determined first. All detection systems were used to analyse the corresponding L1 and L2 specimens, conducting 20 consecutive tests. All tests were completed within one day, 20 S/CO values were observed, the results were judged and the standard deviation and coefficient of variation were calculated. The result was judged to be 100% in line, and the coefficient of variation was less than 10%. Between-run precision was examined second. The detection system analyses the corresponding L1 and L2 specimens, which were performed once per day, continuously detecting for 20 days, observing the S/CO value 20 times, judging the result and calculating the standard deviation and coefficient of variation. The result was judged to be 100% in line, and the coefficient of variation was less than 15%.http://www.R-project.org/).All statistical analyses were conducted using R software  and Youden’s index were used to calculate the optimal diagnostic threshold  of the detection system.Clinical characteristics of all samples are present in To test the precision of each kit, we performed within-run and between-run detections. As seen from A total of 50 patients were considered to have COVID-19 because a viral nucleic acid test appeared positive, and the other 130 controls had negative viral nucleic acid results. Overall, 180 subjects were tested with the COVID-19-specific serological assay. The results showed varying sensitivity and specificity among different kits. D_Ab performed best (92% sensitivity and 99.23% specificity), and B_IgM performed worse than the others .The ROC curve was depicted by using the original S/CO value . AccordiConsidering that endogenous and exogenous factors exist in the process of antibody assays, subgroups of controls, including patients with acquired immune deficiency syndrome (AIDS), tumours, or pregnancies or those older than 90 years old, were involved in the current analysis. Each system had false-positive results in the selected subgroup of controls . It is w11 In the current investigation, the average time of serum collection in all subjects was 13 days after diagnosis; therefore, specific IgM and IgG antibodies should already exist in the specimens.SARS-CoV-2 belongs to the β genus and is the seventh most well-known coronavirus that infects humans. Its nucleocapsid protein (NP) stimulates the human immune system to cause chemical reactions. Specific IgM antibodies emerge on the seventh day of infection and appear to peak after 28 days. Specific IgG antibodies emerge around the 10th day of infection and reach a peak after 49 days, which can be maintained for a long time in the blood. The median time for total plasma antibodies appears on the 12th day after infection.5 the suspected cases are positive for serum SARS-CoV-2-specific IgM/IgG antibodies, are positive for SARS-CoV-2-specific IgG antibodies, or have SARS-CoV-2 IgG antibody concentrations that are four times greater in the recovery period than in the acute period, which can confirm the diagnosis of COVID-19.13 The diagnosis standard of COVID-19 is a changing situation. There is substantial market demand for SARS-CoV-2 antibody detection reagents worldwide. Manufacturers domestically produce antibody detection reagents that are used in the clinical laboratory. Previous investigations have shown that the clinical specificity and sensitivity of some anti-SARS-CoV-2 IgM antibodies are 96.2% and 70.24%, respectively. The clinical specificity and sensitivity of anti-SARS-CoV-2 IgG antibodies are 92.4% and 96.1%, respectively. Therefore, false-negative and false-positive results will appear, which will cause confusion in clinical judgment. Thus, the laboratory needs to pay close attention to the performance indicators of the reagents used.With the published guidelines of the Novel Coronavirus Pneumonia Diagnosis and Treatment Program ,13 the performance indicators of qualitative kits should focus on repeatability, clinical accuracy  and verification of the cut-off value. The results showed that the repeatability of all detection systems is in line with the manufacturer's statement, but the variance among them is relatively large. Specifically, the coefficients of variation regarding B-IgM and B-IgG are larger than those of the others.Seven detection kits from four chemiluminescence systems were used in the current study. All of the kits have been permitted for use via the emergency approval of the China National Drug Administration or the EU CE sales and have been applied to clinical detection. According to the requirements of the People's Republic of China Health Industry Standard WS/T 505–2017 ‘Qualitative Measurement Performance Evaluation Guidelines’,15 In the use of preliminary screening tests, the sensitivity should be greater than 95%. In the case of diagnosis, both the sensitivity and specificity should be greater than 95%. In a confirmed diagnostic test, the specificity should be greater than 98%.12 According to the results of the current study, the clinical sensitivity and specificity of all detection systems do not meet the requirements of screening, diagnosis and confirmation of diagnosis experiments. Therefore, all detection systems cannot be used independently for the diagnosis of SARS-CoV-2 infections and need to be used together with nucleic acid tests and clinical symptoms.According to the WS/T 494–2017 guideline, the sensitivity and specificity of qualitative items for different occasions are also regulated.17 The results of the current investigation showed that B_IgM has the lowest sensitivity, indicating the possibility of more false negatives that can occur during a period of viral infection or in patients with low immunity.Regarding the confounding factors influencing detection results, we divided controls into subgroups that included patients with AIDS, tumours, or pregnancy and older people over 90 years old.18 The reason for false positives may be due to some interfering substances  present in the specimens. Simultaneously, according to the area under the ROC curve of each detection system, the diagnostic accuracy of B_IgM and B_IgG was also the worst, and the diagnostic accuracy of the other systems was better. In addition, according to the ROC curve and Youden’s index, the best diagnostic thresholds were exhibited by A-IgM and C-IgG, and those of the others were inconsistent with the manufacturer's declaration. The optimal thresholds of A_IgG, B_IgM, C_IgM and D_Ab are less than the cut-off value, indicating increased false-positive results. The optimal threshold of C_IgM is greater than the cut-off value, indicating additional false-negative cases.B_IgG has the lowest specificity, which indicates a higher false-positive rate; this situation is prone to occur in special patients, such as those with AIDS, solid tumours, or pregnancy and elderly individuals.Therefore, the laboratory should conduct the necessary performance evaluation of the selected anti-novel coronavirus antibody, carefully interpret the results of the anti-novel coronavirus antibody, perform necessary further testing requirements and reduce missed diagnoses and misdiagnoses.


Water contact angle measurements reveal that the TS-CSRGs are equally hydrophobic to cicada wings, providing them with potential self-cleaning and bactericidal properties. Finite-difference time-domain simulations are used to validate the nanofabrication parameters and to further confirm the polarization-independent electromagnetic field enhancement of the nanostructures. As a real-world application, label-free detection of melamine up to 1 ppm, the maximum concentration of the contaminant in food permitted by the World Health Organization, is demonstrated. The new bioinspired functional TS-CSRG SERS substrate holds great potential as a large-area, label-free SERS-active substrate for medical and biochemical sensing applications.This article introduces a bioinspired, cicada wing-like surface-enhanced Raman scattering (SERS) substrate based on template-stripped crossed surface relief grating (TS-CSRG). The substrate is polarization-independent, has tunable nanofeatures and can be fabricated in a cleanroom-free environment via holographic exposure followed by template-stripping using a UV-curable resin. The bioinspired nanostructures in the substrate are strategically designed to minimize the reflection of light for wavelengths shorter than their periodicity, promoting enhanced plasmonic regions for the Raman excitation wavelength at 632.8 nm over a large area. The grating pitch that enables an effective SERS signal is studied using Rhodamine 6G, with enhancement factors of the order of 1 × 10 Biomimicry is an emerging field with the objective of replicating physical or chemical attributes found in nature to create human-made devices. The development of biomimetic materials and devices has been particularly useful in optics and sensing applications ,2. ExampNanostructures that support surface plasmon resonance (SPR) have been widely used for sensing and biosensing applications through the use of different techniques, including SPR spectroscopy ,23,24,25Here, we present a polarization-independent, template-stripped Ag CSRG (TS-CSRG) SERS substrate, inspired by the tapered nanopillars found in the Cicada wings , along wImaging of the cicada wings, CSRGs, and TS-CSRGs structures was performed using a Dimension Edge atomic force microscope (AFM) system . A ScanAsyst-Air AFM tip  was utilized to scan a 5 µm × 5 µm area, using the peak-force tapping mode, with a scan rate of 1 Hz per line. Bruker NanoScope Analysis software was used to fit and analyze the AFM scans and obtain parameters such as the topography, depth, and pitch of the structures.spk is the surface plasmon wave number, k0 is the incident light wave number in free space, n is the refraction index of the dielectric, θ is the incidence angle, m is the diffraction order , and Λ is the grating pitch.The surface plasmon resonance wavelength of CSRGs is found by matching the SPR wavevector to the diffracted light via the grating equation so that the following equality is obtained :ksp = k02) was directed onto a Lloyd mirror optical setup to allow for molecular mass transport of the azo-molecules to generate of nanopatterned SRGs. After the initial inscription of the SRGs (time of exposure = 300 s), the sample was rotated by 90° and a second exposure for 100 s was performed to fabricate orthogonally superimposed SRGs. An 80-nm layer of silver was subsequently sputtered over the CSRG using a Bal-Tec SCD 050 sputter-coater, to make an Ag-CSRG. The prepared samples had a periodicity of 450, 500, 550, 600 nm.Fabrication of the CSRGs was performed using the rapid and high-throughput interferometric technique described elsewhere ,21,26. AThe fabricated Ag-CSRG was spin-coated with a UV-curable epoxy  to generate uniform epoxy coating. Next, a pre-cleaned Corning 0215 soda lime microscope glass slide  was pressed against the epoxy-coated Ag-CSRG. The sandwiched system was then exposed to UV light in an enclosed UV chamber  for 30 min. When the epoxy was cured, the patterned silver was stripped from the Ag-CSRG by a simple peel-off. The stripped substrate, consisting of the smooth Ag nanogratings, was subjected to a final rinsing with 10% ethanol and DI water to dissolve and remove any remaining gDR1 from the metal surface. The cleaned substrate was then air-dried and stored in a microscope glass slide holder for further use.A Horiba/Jobin-Yvon Raman spectrometer (Model: LabRAM) with a 632.8 nm HeNe laser (17 mW), 1800 1/mm grating and an Olympus BX-41 microscope system were used. The collection of spectra was performed in the backscattered mode under the following conditions: ×100 microscope objective, 500 μm pinhole, 500 μm slit width, laser filter 10×, for a sampling time of 10 seconds with 10 repeats. All Raman spectra were background corrected through polynomial subtraction, and the noise was reduced with a Savitsky–Golay filter.−1 (1000 ppm) and diluted in water to generate solutions in the range of 1–100 ppm.R6G was dissolved in methanol at a stock concentration of 0.1 M and diluted in methanol to generate solutions in the range of 1 µM–1 mM. Melamine was dissolved in Millipore® water to a stock concentration of 1 mg mLContact angle measurements were performed using an OCA 15EC digital goniometer . Droplets  of Nanopure water were dispensed onto a 500 nm Ag TS-CSRG at standard conditions using an electronically controlled syringe. The resulting contact angle was calculated using the SCA 20 software module  with a Young-Laplace fitting feature for the sessile drop method.SERS EF for R6G molecule absorbed on Ag-CSRG was calculated using the following equation :EF = , respectively. BulkN is the number of molecules illuminated in bulk, giving a normal Raman signal, and SERSN is the number of molecules illuminated on the nanostructured metallic substrate, giving the SERS signal. The peak at 1358 cm−1 represents intensity at a characteristic band wave number for R6G absorbed on an Ag-CSRG and a flat Ag substrate.A and p correspond to the amplitude and period of the structure, respectively, in accordance with the AFM characterization. A uniform mesh size of 3 nm was used for the envelope of the nanostructure, comprising the UV-curable epoxy, the silver film, and the dielectric medium in all the directional axes. A time-averaged electric field intensity distribution, normalized with respect to the incident plane wave |E/E0|2, was calculated for the Ag-CSRG. A frequency-domain field profile is placed at the xy plane of the CSRG. To match experimental conditions, |E/E0|2 was recorded at 632.8 nm, corresponding to the excitation wavelength of the Raman apparatus.Three-dimensional FDTD was used to simulate the distribution of the near-field electromagnetic field on the surface of the TS-CSRG using Lumerical FDTD Solutions software. Simulations under S and P polarizations were recorded and added to emulate the plasmonic response under a quasi-unpolarized broadband excitation light source. Symmetric and antisymmetric boundary conditions were set for the x- and y-directions, respectively, and a perfectly matched layer (PML) in the z-direction. The dielectric permittivity used in the simulations for the UV-curable epoxy and silver were obtained from the manufacturer and the literature, respectively . The topHigh-magnification image acquisition of the surface of TS-CSRGs was achieved using a high vacuum scanning electron microscope (SEM) Quanta FEG 150 ESEM  with BF/DF STEM detector, at 10 kV. Images of TS-CSRG of 450, 500, 550 and 600 nm were acquired at magnifications of 16,000×, 20,000× and 25,000× . Cryptotympana atrata fabricius reported previously [A piece of the external façade of the wing of a natural cicada eviously .Cryptotympana atrata fabricius. The nanostructures have a total sensing area of approximately ~1 cm2, allowing for a large-area approach for target analyte detection, in contrast to established hot spot methods.As the nanostructure pattern in the cicada wing is nearly complementary to a CSRG, it can be reproduced via template-stripping to create the bioinspired SERS-active substrate. The fabrication procedure of the TS-CSRG is schematically shown in Neotibicen canicularis wing and atop a pristine TS-CSRG taken with the automatic CA measurement system. From the images, it is qualitatively evident the hydrophobicity exhibited by both surfaces. Quantitatively, the measured CA from the cicada wing and the TS-CSRG were, respectively 115° ± 2.075° and 119° ± 3.4222° (n = 5).The nanostructures on the wings of the cicadas not only provide them antireflection but also self-cleaning and antibacterial properties that arise from the hydrophobicity of the surface . CompareElectromagnetic enhancement is critical for SERS-based detection. In a backscattering approach, a surface-confined enhancement assisted by SPR excitations allows for the enhancement of small molecule Raman signals. However, a SERS substrate needs to be tailored to allow for the excitation depending on the incident laser wavelength. Using Equation (1), for air (n = 1) and assuming normal incidence, the desired pitch of the gratings was calculated to be ~560 nm for a laser excitation wavelength of 632.8 nm. However, any analyte on the surface of the metal will eventually change the dielectric permittivity as perceived by the incident light. Hence, a set of TS-CSRGs with a grating pitch ranging from 450 to 600 nm with a 50-nm pitch increment was fabricated to acknowledge the dielectric change encountered by the incident light on the surface. The nanostructures were strategically designed with those periodicities to minimize the reflection of light for wavelengths shorter than the periodicity, promoting therefore enhanced plasmonic regions for the Raman excitation wavelength of 632.8 nm.FDTD simulations were used to demonstrate the polarization-independent electric field enhancement in the vicinity of the TS-CSRG and to confirm the nanostructure pitch leading to the highest EF. Details on the methodology, including the equation utilized to replicate the topography of the nanostructures, are described in the Experimental section. E/E0|2, recorded along the xy cross-section, for gratings with periodicities spanning from 450 nm to 600 nm for a dielectric with RI of 1.33. All the simulation results were scale-adjusted for intensity values of 0–100. The plasmonic enhancement obtained for all the structures demonstrated to be the same for s- and p-polarized incident light. −1 are characteristic of R6G [−1, corresponding to aromatic C-C stretching [−1 was used for the EF calculations, and the corresponding intensity for R6G (10−2 M) on a flat Ag substrate was calculated to be 70 arbitrary units (a.u.). The intensities at 1358 cm−1 were recorded for each of the substrates with different pitches using R6G (10–5 M). The EF values were calculated using Equation (2) for TS-CSRG with pitches 450, 500, 550 and 600 nm were 3.8 × 104, 7.6 × 104, 6.1 × 104 and 1.6 × 104, respectively. The TS-CSRG with a pitch of 500 nm exhibited the highest EF—a value that may serve as a guideline for SERS detection applications and further investigations, with magnitude comparable to reported values for grating-based SERS substrates [The pitch-dependency of the TS-CSRG was used to tailor the SERS signals response upon the adsorption of Rhodamine 6G (R6G), a Raman reporter molecule with a distinct Raman spectrum. c of R6G ,52. The retching , is commbstrates  and commbstrates .−5 M) as the maximum residue limit for melamine in milk and 1 ppm (7.9 × 10−6 M) as the maximum residue limit in infant formula [−1), depending on reaction conditions [The TS-CSRG was further evaluated in a real-world detection scenario for the detection of melamine. Melamine is a toxic, nitrogen-rich (66% by mass) chemical used in the plastics industry for the production of compounds for molding, coating, adhesives, and glues. Due to its high nitrogen content, it is illegally added to foodstuffs such as pet food, milk, infant formula to inflate the apparent protein content of the food . Melamin formula . Detectinditions . This prCryptotympana atrata fabricius and are equally hydrophobic, providing them with potential self-cleaning and bactericidal properties. The nanostructures enable a field enhancement that allows for the sensitive and reproducible SERS detection of R6G. Simulations and experimental investigation of the SPR-assisted electromagnetic enhancement are performed via FDTD and detection of SERS-active dye R6G, respectively, to validate the nanofabrication parameters. More important, the TS-CSRG enables the label-free, sensitive detection of melamine at concentrations compatible with the maximum residue limits allowed by the WHO in food. The fabrication methodology of TS-CSRG allows for the generation of nanostructures with customized periodicities that can be tailored for specific applications. Therefore, the new bio-inspired functional, SERS-active TS-CSRG introduced here holds great promise as large-area, label-free SERS-active substrates for medical and biochemical sensing applications.In conclusion, this work presents a new Ag TS-CSRG as polarization-independent SERS active substrate, inspired by the tapered nanopillars found in cicada wings. The fabrication of the substrate is cost-effective and achieved in a cleanroom-free environment via holographic exposure followed by a template-stripping step using a UV-curable resin. Inspired by the AR properties of the cicada wings, the nanostructures are strategically designed to minimize the reflection of light for wavelengths smaller than their periodicity, promoting enhanced plasmonic regions for the Raman excitation wavelength at 632.8 nm. AFM scans reveal that the TS-CSRGs possess a remarkable resemblance to the nanostructures in the wings of cicada


Hypoxia is one of the important properties of solid tumor. However, oxygen supply within tumors is generally heterogeneous according to the distance from the nearest blood vessel. The discrepancy of metastatic potential exists between hypoxic cancer cells and relatively normoxic cancer cells. But the molecular mechanism remains poorly understood.Methods: Differential expression of circRNAs in plasma exosomes of CRC patients and normal subjects was performed by screening. Exosomes were isolated by ultra-centrifugation and RNA expressions were determined by RT-qPCR. The migratory capacity of cells was performed by high intension imaging, wound healing assay and transwell chamber migration assay.Results: Circ-133 is enriched in the plasma exosomes of CRC patients and increased with the disease progression. Exosomal circ-133 derived from hypoxic cells delivered into normoxic cells and promoted cancer metastasis by acting on miR-133a/GEF-H1/RhoA axis. Meanwhile, animal experiments revealed that knockdown of circ-133 can inhibit tumor metastasis. Circ-133 is expected to be a new biomarker for monitoring tumor progression and might be a novel therapeutic target.Conclusions: Hypoxia-derived exosomal circ-133 transported into normaxic cancer cells and promoted cell migration via miR-133a/GEF-H1/RhoA axis. This study reveals a potential mechanism for that the intra-tumor heterogeneity of oxygen promote cancer progression. On the basis of the estimated results in 2018, CRC has become the second leading causes of cancer-related death, and the incidence of it has ranked the third for both sexes Hypoxic microenvironment is a substantive characteristic of solid tumor which results from rapid proliferation of cancer cells plus abnormal structure and function of tumor blood vessels Circular RNAs (circRNAs) are a kind of endogenous RNA formed by alternative splicing, and are widely distributed in eukaryotic cells. Owing to its closed ring structure, circRNAs are not easily degraded by endonuclease and are more stable than linear RNA Metastasis is a multistep complex process that be restrained by an intricate regulation network In the case of insufficient oxygen supply, a vast majority of the adaptive reprogramming in cancer cells are driven by HIF-1 protein CRC tissues and paired adjacent noncancerous tissues were obtained from patients undergoing surgical procedures at the Tianjin Medical University Cancer Institute and Hospital . Both the tumor tissues and noncancerous tissues were histologically confirmed. Written consent was provided by all of the patients, and all aspects of this study were approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. Tissue fragments were immediately frozen in liquid nitrogen at the time of surgery and stored at -80 °C.Female nude mice  were purchased from the Model Animal Center of Nanjing University, fed in an SPF (specific pathogen-free) animal facility. All of the experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee of Tianjin Medical University Cancer Institute and Hospital.2. Hypoxia studies were carried out at 1% oxygen.Human colorectal cancer cell lines HCT116 and SW480 were purchased from the Shanghai Institute of Cell Biology of the Chinese Academy of Sciences . HCT116 were cultured in 1640 , and SW480 were cultured in L15 medium . Both of the basal culture media above were supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin. Cells were incubated in a humidified incubator at 37 °C with 5% COExosomes were isolated from cell culture medium and plasma by sequential differential centrifugation, according to previous publications 3). The samples were dehydrated in increasing concentrations of alcohol . Pure alcohol was then exchanged for propylene oxide, and specimens were infiltrated with increasing concentrations of Quetol-812 epoxy resin mixed with propylene oxide. Samples were embedded in pure, fresh Quetol-812 epoxy resin. Ultrathin sections (100 nm) were cut using a Leica UC6 ultramicrotome and were poststained with uranyl acetate and lead citrate for 5 min at RT before observation via a FEI Tecnai T20 transmission electron microscope operated at 120 kV.For conventional transmission electron microscopy, the exosome pellet was placed in a droplet of 2.5% glutaraldehyde and fixed overnight at 4 °C. Then, the sample was post fixed in 1% osmium tetroxide at room temperature (RT), embedded in 10% gelatin, fixed in glutaraldehyde at 4 °C, and cut into several blocks (<1 mm-6 M) was prepared by adding 0.4 µl of PKH26 ethanolic dye solution to 100 µl of Diluent C. The 100 µl of exosome suspension was then mixed with the 100 µl of dye solution by pipetting. After incubating the cell/dye suspension for 1-5 min with periodic mixing, the staining was stopped by adding 200 µl of serum and incubating for 1 minute. The stained exosomes were finally washed twice with 1× PBS and were resuspended in a fresh sterile conical polypropylene tube.PKH26 Red Fluorescent Cell Linker Kits (Sigma) were used for lipid bilayer labeling. Exosomes were first resuspended in 100 µl of Diluent C. A dye solution (4 ×10HCT116 and SW480 were seeded into 6-well plates and transfected using Lipofectamine 2000 (Invitrogen) and Opti-MEM  according to the manufacturers' instructions. For circ-133/miRNA upregulation and downregulation, a 100 pmol dose of circ-133-plasmid, si-circ-133, miR-133a mimics, inhibitors and negative control (NC) were used. In addition, HCT116 and SW480 were harvested 24 h or 48 h after transfection to isolate total RNA or total cell lysate. HCT116 and SW480 cells were cultured in 100 mm dishes and were transfected with si-circ-133 and NC , and the culture medium was replaced with 1640/L15  with exosome-free FBS  for the isolation of exosomes.Total RNA was extracted from the cultured cells and tissues using TRIzol reagent (Invitrogen) according to the manufacturer's protocols. We adopted TaqMan microRNA probes  to quantitate miRNA. After the reactions were complete, the cycle threshold (CT) data were determined using fixed threshold settings, and the mean CT values were determined from triplicate PCRs. We used the formula to calculate the relative quantities of target genes. U6 snRNA was used as an internal control for the miRNAs; β-actin, for the circ-133 and GEF-H1 mRNA levels. The β-actin, GEF-H1 and circ-133 primers were designed as follows:5'-TCTATCCTGTGCTCTACCCCA-3' ;5'-TGAGAAGACAAGGTGGCCGAG-3' ;5'-TCCCTCATTGACGAAGCAGA-3' ;5'-GGTGCAGCTCTGTCTGGATT-3' ;5'-TCGAGGTGGATGGAAAGCAG-3' ;5'-CACAAGACAAGGCACCCAGA-3' ;5'-GGCTGTGCTATCCCTGTACG-3' ;5'-CTTGATCTTCATTGTGCTGGGTG-3' .The expression of protein was assessed by western blot analysis, and its expression in the samples was normalized to β-actin expression. Cells and tissues were lysed in RIPA buffer with freshly added protease inhibitor cocktail. Total lysates were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore). The immunoblots were blocked with 5% BSA at room temperature for 1 h and incubated at 4 °C overnight with anti-HIF-1α , anti-GEF-H1 , anti-RhoA , anti-E-cadherin , anti- β-actin , anti-CD63 , and anti-TSG101 . After incubation with the secondary antibody, the membranes were visualized with an enhanced chemiluminescence system kit  according to the manufacturer's protocol.http://www.targetscan.org/), PicTar (http://pictar.mdc-berlin.de/) and miRanda (http://www.microrna.org/). The reporter plasmid p-MIR-circ-133, p-MIR-GEF-H1 and p-MIR-RhoA was designed by Genscript . For the luciferase reporter assays, 2 mg of firefly luciferase reporter plasmid; 2 mg of β-galactosidase vector; and equal doses (200 pmol) of mimics, inhibitors, or scrambled NC RNA were transfected into cells. The β-galactosidase vector was used as a transfection control. At 24 h after transfection, cells were analyzed using a Dual Luciferase Assay Kit (Promega).The miRNA target prediction and analysis were performed with the algorithms from TargetScan (5 cells per 8.0 µm pore size polycarbonate membrane insert). In addition, 500 µl of complete media was added to the lower compartment. After incubation for 18-24 h, non-migrated cells on the upper surface of the insert were removed gently by cotton swabs, and cells that migrated to the bottom of the membrane were fixed and stained using a Three-Step Stain Set (Thermo). The number of invaded cells was counted under a light microscope. To minimize the bias, cells in five randomly selected fields at a 200× magnification were counted to calculate the average cell number.High intension imaging , wound healing assay and Transwell chamber  migration assay was used to determine the migratory capacity of SW480 and HCT116. For the wound healing test, cells were seeded in 6-well plates. In addition, 24 h later, each well was scraped with a 20 µl pipette tip to create 2 linear regions devoid of cells, and medium without FBS was added. Then, at the time points of 0 h, 12 h and 24 h, cells were observed and photographs were taken. For the Transwell chamber test, the pretreated cells were transferred into the upper chamber in 200 µl of serum-free growth medium . Cell suspension diluted with PBS to a concentration of 1 million/ml. After repeated freezing and thawing, the cell rupture and release of intracellular components. Centrifuge at 2000-3000 rpm for 20 minutes at RT, carefully collected the supernatant. Then follow the instructions for subsequent enzyme labeling, incubation and color rendering. Finally, OD value at 450 nm was analyzed by microplate reader.Dynabeads™ Streptavidin Trial Kit  was used to complete the experiment. Biotinylated miR-133a and its corresponding negative controls were transfected into 293T cells respectively. After 24 hours of culture, the cells were lysed. And the cell lysates were co-incubated with the streptavidin beads. The beads are eluted with the aid of the DynaMag™ Magnet. Finally, PCR was performed for eluting complexes to analyze the abundance of circ-133, GEF-H1 mRNA and RhoA mRNA.6 cells in 0.2 mL PBS per mouse, 6 mice per group). After a week, Exosomal-OE.circ-133 or Exosomal-KD.circ-133 were injected into caudal vein every three days. Mice were sacrificed 28 days after injection to remove the xenografted tumors, and the volumes and weights of the tumors were recorded. The tumor was divided into two parts; one part was used for protein and total RNA extraction, and the remaining tissue was used for immunohistochemical staining for GEF-H1 and E-cadherin. CTCs were detected by were detected by flow cytometry.The lentiviral expression plasmids were bought from Shanghai Genechem Co. Ltd. Puromycin  was used to successfully infect HCT116 cells and obtain stable high expression of GEF-H1, low expression of GEF-H1 and control HCT116 cells. Then, those cells were inoculated subcutaneously into SCID mice  tail. Recent studies have demonstrated that it can be secreted into the extracellular environment by exosomes to mediate intercellular communication. We isolated plasma exosomes from CRC patients and normal subjects, and these exosomes were firstly morphologically identified by transmission electron microscopy, and the exosomal markers Tsg101 and CD63 were also detected Figure F. As is To determine the expression of circ-133, qRT-PCR was performed in 17 normal subjects and 25 CRC patients A. Plasmain vitro experiments. SW480 and HCT116 were cultured under hypoxic and normoxic conditions respectively. Culture medium was collected and exosomes were extracted to detect the level of circ-133. The results showed that exosomes derived from hypoxic cells were rich in circ-133, which gradually increased with the prolongation of hypoxic time (Figure Circ-133 expression was higher in CRC cell lines than in normal colonic epithelium cells NCM 460 C. And thIn our study, exosomes were isolated by sequential differential centrifugation, morphological identification by transmission electron microscopy, and specific markers detection by western blotting Figure A. As shoIn this study, high intension imaging cell analysis, transwell assay and wound healing test were combined to analyze changes in cell migration capacity. As shown in Figure The abundance of circ-133 in SW480 and HCT116 cells were directly changed by transfection, including four groups: OE. circ-133, OE. NC, si-circ-133 and si-NC. High intension imaging cell analysis showed that the motility of cells in OE. circ-133 group enhanced, while that in si-circ-133 group reduced (Figure Meanwhile, the expression of GEF-H1 and RhoA was up-regulated and E-cadherin was reduced in cells of the OE. circ-133 group, while that of the si-circ-133 group was reversed Figure A. The rein vitro, that hypoxic cells-derived exosomal circ-133 were transported into relative normoxic cells and then targeted GEF-H1/RhoA by adsorbing miR-133a, thereby reducing the distribution of E-cadherin on the membrane, enhancing the migration ability of cancer cells (Figure Thus, our hypothesis was confirmed s Figure D.in vivo. Figure The effect of the circ-133/GEF-H1/RhoA axis on the metastatic potential of colorectal cancer was then further validated CRC is still a serious threat to human health due to its high incidence and mortality. Improvements in physical examination and guidelines have made it possible to curb the development of CRC at an early stage. But a certain percentage of patients are still diagnosed at an advanced stage Microenvironmental hypoxia is an important intrinsic property in the rapidly growing solid tumor The regulation of miRNAs to genes at the post-transcriptional level is not one-to-one, but presents a complex network regulation pattern E-cadherin is a pivotal molecule that mediates the connection between epithelial cells, and its reduced distribution on the membrane is a key initial link for the invasion and metastasis of cancer cells in vitro and in vivo that exosomal circ-133 derived from hypoxic cells transported into normoxic cells and regulated the E-cadherin membrane distribution, promoting cancer metastasis via miR-133a/GEF-H1/RhoA axis.In this study, a group of differentially expressed circRNAs were screened from the plasma exosomes of CRC patients and normal subjects. circ-133 was selected, which is related to hypoxia and regulates the distribution of E-cadherin protein across cell membrane. Then it was verified both Circ-133 was enriched in the plasma exosomes of CRC patients and gradually increased with disease progression, suggesting that exosomal circ-133 is expected to be biomarker for monitoring the progression of CRC. In addition, animal experiments revealed that reducing the level of circ-133 can weaken the metastatic potential of CRC, which is a potential new therapeutic target.In summary, our study is the first to explore the role of intra-CRC oxygen supply heterogeneity in promoting tumor metastasis. And the intercellular signaling pathway between hypoxic and normoxic cells mediated by exosomes has been revealed for the first time from the perspective of epigenetic regulation. However, there are many components in exosomes, and the carcinogenic effect of heterogeneous oxygen supply is complex, which still needs further study in the future.Supplementary figures and tables.Click here for additional data file.


There was no difference among groups containing ZnO-NN regarding their flow. The ZnO-NN addition significantly increased the film thickness. Water sorption and solubility tests showed no difference among groups. The radiopacity increased, and DC decreased with higher concentrations of ZnO-NN. Micro-Raman suggested that ZnO-NN was in close contact with root canal dentin. Overall, the incorporation of ZnO-NN provided an antibacterial effect against Enterococcus faecalis without a significant detrimental impact on the physical and chemical functionality of the material. The use of ZnO-NN as an inorganic filler is a potential application within dental materials intended for root canal treatment.This study aimed to evaluate the effect of needle-like zinc oxide nanostructures (ZnO-NN) on the physical, chemical, and antibacterial properties of experimental methacrylate-based dental sealers. ZnO-NN was synthesized and characterized. ZnO-NN was added to a co-monomer blend at 20, 30, and 40 wt.%. One group without ZnO-NN was used as a control. The dental resin sealers were evaluated for their flow, film thickness, water sorption, solubility, radiopacity, degree of conversion (DC), dental-sealer interface characterization via micro-Raman, and antibacterial activity. ZnO-NN presented a mean needle diameter of 40 nm and 16 m Nanomaterials have received widespread attention for dental applications partly because they exhibit improved or new functionality in comparison to their microsized counterparts ,2,3. In As biointeractive fillers, nanosized ZnO particles can enable mineral growth via bioactivity and negatively affect metalloproteinases and bacteria growth ,9,10. PaOver the past decade, the synthesis, characterization, and potential biomedical applications of one-dimensional nanostructure ZnO have been studied ,14. A laEnterococcus faecalis, an essential bacterium in the post-infection of root canal treated teeth, and failure of treatment [The antibacterial performance of ZnO nanostructures depends on their size and shape, surface-to-volume ratio, and the number of oxygen vacancy sites ,19. ZnO reatment . This anreatment ,21. Thesreatment ,23. Nanostructured needle-like zinc oxide (ZnO-NN) presents extensions originating from one nucleation core with distinct directions within a three-dimensional space . This stE. faecalis.Based on the above consideration, this study reports the synthesis of needle-like zinc oxide nanostructures (ZnO-NN) particles and their incorporation into an experimental methacrylate-based dental sealer for root canal treatment for the first time. This study aimed to assist the investigators in determining whether a candidate ZnO nanostructure has scientific merit to justify further investigations and applicability. For this purpose, we evaluated the dental sealers for the following physical, chemical, or biological properties: flow, film thickness, water sorption, and solubility, radiopacity, degree of conversion, root-sealer interface, and antibacterial effects against Needle-like zinc oxide nanostructures were prepared by a simple thermal evaporation method, according to a previous study . Briefly−2, Radii; SDI, Bayswater, Australia) was used to photoactivate the sealers along with all study.The experimental dental sealers were formulated using urethane dimethacrylate (UDMA), glycerol-1,3-dimethacrylate (GDMA), ethoxylated bisphenol A glycol dimethacrylate (BISEMA), camphorquinone (CQ), N, N-dihydroxy ethyl-para–toluidine (DHEPT) and benzoyl peroxide (BP). These materials were used without further processing. The flow test was conducted in accordance with ISO 6876:2012 . A totalThe film thickness evaluation was conducted following the ISO 6876:2012 . Two glam1) was obtained . After final drying, two measurements of the diameter, at right angles to each other, were taken with a digital caliper, and the mean diameter was calculated. The thickness of the specimen was measured at the center of the sample and at four equally spaced points on the circumference. The area was calculated, in mm2, from the mean diameter, and then, using the mean thickness, the volume was calculated in mm3. Water sorption and solubility tests were performed according to ISO 4049:2009 , except per sample. Afterward, the samples were removed from the liquids, which were stored in a light-free container. Each specimen was weighed after being dried slightly to blot away the surface water, and the weight was obtained (m2), i.e., the mass of the hydrated samples. After weighing, the samples were returned to the first desiccator. The cycle was repeated until the constant weight was recorded as m3. Water sorption (WS) was calculated according to Equation (1), and the solubility (SL) according to Equation (2) [The samples were stored in water at 37 °C for seven days; the volume of water for immersion was 10 mL tion (2) .(1)WS= was directly dispensed (3 µL) on the top of the diamond crystal and photoactivated for 60 s (n = 3). Data were evaluated with the Opus software , with Blackman–Harris 3-Term apodization in a range of 4000 to 400 cm−1 and a resolution of 4 cm−1. The DC was calculated based on the intensity corresponding to the stretching of the carbon–carbon double bond (peak height) at 1635 cm−1, and a symmetric ring stretching at 1610 cm−1 of non-polymerized and polymerized samples [The degree of conversion (DC) was measured by Fourier Transform Infrared Spectroscopy (FTIR) with a spectrometer  equipped with an attenuated total reflectance device, composed of a horizontal diamond crystal with a mirror angle of 45 ° . A light samples . The anaFour lower incisor human teeth were cleaned of organic debris and stored in distilled water at 4 °C. The roots were sectioned below the enamel cement junction to obtain roots with a final length of 15 mm. The root canals were chemical-mechanically prepared with first series Kerr files until 40 files, in an ascending sequence, using a step-back technique, associated with sodium hypochlorite 1% irrigation. The canals were prepared 1 mm below the final strength. After the chemical mechanical preparation, the canals were irrigated with 3 mL EDTA for 1 minute and then washed with 3 mL of sodium hypochlorite and 3 mL of distilled water. The canals were dried with absorbent paper cones. The roots were then randomly divided and filled with the experimental sealer and gutta-percha cones as core material. The sealer was photoactivated for 40 s from the top of the root canal cervical portion and stored at 37 ± 1 °C for seven days. −1, and spectra were analyzed between 80 and 1525 cm−1. After this period, the roots were sectioned at low speed under constant irrigation  perpendicular to the long axis of the root. A scan in mapping mode by Raman vibrational spectroscopy (micro Raman) was performed in line with 50 µm in each slice, including sealer and dentin, so it was possible to characterize the interface and analyzed the sealer penetration into dentin. Micro-Raman spectroscopy was performed using a SENTERRA Raman Microscope . It was used 785 nm laser for 10 s with five co-additions, totaling 60 s with 100 mW of laser power, resolution of 3–5 cmEnterococcus faecalis, a collection strain of this bacterium  was used in this study. Bacteria from frozen stock cultures were grown aerobically in Brain Heart Infusion (BHI) broth  at 37 °C. Cells were harvested by centrifugation and resuspended in fresh medium. The inoculum was prepared by adjusting the cell suspension to a predetermined optical density (OD) of 0.2 at 600 nm.To evaluate the antibacterial effect of the dental sealers against E. faecalis suspension. The negative control consisted of three sets of wells containing uninoculated fresh medium (300 µL). For direct contact inhibition, three samples per group were produced with 3 mm (±0.1 mm) in diameter and 1.0 mm (±0.1 mm) thick. The samples were sterilized in hydrogen peroxide plasma. Using a 96-well flat-bottom plate, each specimen was placed on a well containing 300 µL BHI broth . Then, each well was inoculated with 20 µL of the −8. The 10−1, 10−3, 10−6, and 10−8 dilutions were plated in BHI Agar (HiMedia Laboratories), using 25 µL aliquots of each dilution in duplicate. Plates were incubated at 37 °C under anaerobic conditions. After 24 h, the colonies were counted visually, scaled by dilution factors, and then expressed as colony-forming units (CFUs) per milliliters. The groups were statistically compared to each other. The experiment was carried out under aseptic conditions.Immediately after the placement of inoculums and after 24 h, 90 µL of each well content was diluted in saline to 10Data of radiopacity, flow, film thickness, water sorption, and solubility were analyzed using one-way ANOVA and Tukey. The antibacterial analysis was analyzed via two-way ANOVA (concentration of ZnO-NN and time) and the Student Newman Keuls test. Repeated-measures ANOVA and Tukey analyzed the DC data. All analyses were performed at 0.05 level of significance.p > 0.05). The film thickness ranged from 26.7 (±5.0) mm for 0 wt.% to 56.7 (±5.8) mm for 40 wt.% of ZnO-NN. The groups containing ZnO-NN showed higher film thickness in comparison to 0 wt.% (p < 0.05) without statistical difference among the three concentrations of ZnO-NN (p > 0.5).The flow and film thickness values are displayed in p > 0.05). The radiopacity of the dental sealers ranged from 66.8 (±5.3) pixels to 0 wt.% to 123.2 (±9.0) pixels to 40 wt.% of ZnO-NN. By increasing the concentration of ZnO-NN addition, the higher the radiopacity, with no differences comparing 30 and 40 wt.% of ZnO-NN (p > 0.05). The group containing 40 wt.% of ZnO-NN showed no difference from the 1 mm of aluminum (p > 0.05).p > 0.05). Over time, the group containing 40 wt.% of ZnO-NN increased its DC from 2.4 (±1.2) % to 25.9 (±3.4) % after 14 days of storage (p < 0.05).p > 0.05). After 24 h of contact with the sealers, the groups with ZnO-NN addition showed a reduced number of bacterial viability when compared to the group without ZnO-NN (p < 0.05). At this period, the results ranged from 9.2 (±0.0) log10CFU for 0 % of ZnO-NN to 8.3 (±0.2) log10CFU for the group with 20 wt.% of ZnO-NN (p < 0.05). The groups with 20 to 40 wt.% of ZnO-NN showed no differences among them (p > 0.05).E. faecalis.In the present study, needle-like zinc oxide nanostructures (ZnO-NN) were explored as inorganic fillers to improve dental resin sealers. ZnO-NN was successfully synthesized and incorporated in an experimental methacrylate-based root canal sealer. ZnO-NN addition to the sealer increased its radiopacity, maintaining reliable film thickness, flow, pH, and proving activity against 2/g of surface area in this study, decreasing the volume fraction that could be added to the polymeric matrix. In the present study, nanosized particles (mean needle diameter of 40 nm) of ZnO were produced and used for sealer formulation. The ZnO-NN addition adjusted the viscosity of the sealer, considering that the group without nanoparticle addition showed a flow that could not be measured . One could think that a sealer should present a low viscosity; however, sealers with a high flow could overflow beyond the apex leading to biocompatibility concerns. The ZnO-NN addition improved the handling of the material to be used as an endodontic sealer.The success of endodontic therapy depends on a complete root canal filling to promote adequate sealing . The seap > 0.05). The values found for flow and film thickness were similar to the ones established at ISO 6876:2012 [Despite better handling, the materials proposed with ZnO-NN showed flow above 17 mm even with the addition of 40 wt.%, which is following the requirements of ISO 6876:2012 . The fil876:2012  . Moreover, with 40 wt.% of ZnO-NN, the sealers achieved radiopacity compatible with 1 mm of aluminum. Therefore, further modification in the sealer’s composition via the incorporation of radiopacifying agents, i.e., bismuth oxide or ytterbium trifluoride [Another relevant aspect for sealers is the achievement of radiopacity to make possible the differentiation of the material from the adjacent anatomical structures . ISO 687fluoride , could bThe micro-Raman spectra of the sealer/dentin interface suggested the penetration of ZnO-NN into the root canal dentin. The presence of inorganic filler into the dentinal tubules may improve the stability of dentin-methacrylate material interface . BesidesE. faecalis growth assessed by direct contact inhibition.It is already well established that even after the root canal preparation for endodontic treatment with complete irrigation and shaping, there are still microorganisms on the root canal walls . The imp2O2) and hydroxyl radicals. The reactive oxygen species (ROS) might trigger membrane lipid peroxidation and cause an antibacterial effect.In the literature, the mechanisms of antibacterial activity of nanostructured ZnO are not well understood, although some statements were proposed, such as the generation of hydrogen peroxide could be the main factor of antibacterial activity . Tiwari’2 [2 levels that exceed those of their native habitats [E. faecalis represents an important clinical problem due to virulence factors such as the regulation of the oxidative stress response by transcriptional regulation by estrogen receptors [E. faecalis confer protection against hydrogen peroxide, hydroxyl radicals, and superoxide. The defense against other reactive oxygen or nitrogen species is indirect or nonenzymatic.Some microorganisms escape oxidative stress by living in anaerobic microhabitats; all others must deal with the consequences of intracellular O2 . Howeverhabitats . E. faececeptors . A numbeE. faecalis is a recognized pathogen in post-treatment endodontic infections, and it is considered the most resistant species against chemomechanical preparation [E. faecalis due to the reasons described above. Here, we have estimated the antibacterial activity of sealers containing ZnO-NN using a relevant single-species model. The clinical predictive value of in vitro tests is currently often limited. In this study, even though we observed statistical difference in comparison to 0 wt.% of ZnO-NN, there was no substantial log reduction. The reason for that may be because the test was conducted against planktonic bacteria. Therefore, the leaching of more ZnO-NN to the broth containing E. faecalis would be necessary to increase the differences. After 24 h, the material was probably stable enough not to provide a high quantity of free Zn+.The use of sealer exhibiting antibacterial properties may be useful to decrease or avoid the growth of the remaining microorganisms in the endodontic system . E. faecparation . ConsideFor this reason, further studies could be carried out to understand the effects of the sealers containing ZnO-NN against multi-species biofilm over time. These investigations can be a promising next step to translate the experimental dental sealers containing ZnO-NN into clinical settings.In this work, we explored the applicability of ZnO-NN incorporated in an experimental methacrylate-based root canal sealer to impart improved properties. Overall, the incorporation of ZnO-NN provided an antibacterial effect without a significant detrimental impact on the physical and chemical functionality of the material. The use of ZnO-NN as inorganic filler is a potential application within dental materials intended for root canal treatment.


Periodontitis is one of the most common oral diseases and is a potential risk factor for systemic diseases. In this study, we aimed to investigate the association between periodontitis and learning and memory impairment.Porphyromonas gingivalis lipopolysaccharide  into the palatal gingival sulcus of the maxillary first molars of 10-week-old male rats for a 10-week period. We assessed alveolar bone resorption using micro–computed tomography analysis and learning and memory ability using the Morris water maze test. We determined the levels of cytokines  and LPS in the peripheral blood and cortex, as well as toll-like receptor 4 (TLR4)/NF-κB signaling pathway activation, using reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot. We determined activation of microglia and astrocytes, expression of Aβ1-42, APP and Tau by immunohistochemistry. Finally, we measured the expression of amyloid precursor protein (APP) and its key secretases, as well as the Aβ1-40/1-42 ratio, by RT-PCR, western blot, and ELISA.We established a periodontitis model by topical application of We found that periodontitis induced learning and memory impairment in the rats. Further, we observed that it induced significant alveolar bone resorption. There was an increase in the levels of inflammatory cytokines and LPS. Moreover, we confirmed TLR4/NF-κB signaling pathway activation. We also observed activated microglia and astrocytes with enlarged cell bodies and irregular protrusions. Finally, we observed the promotion of β- and γ-secretases APP processing.Our findings indicated that periodontitis was associated with learning and memory impairment, probably induced by neuroinflammation via activating the TLR4/NF-κB signaling pathway. Furthermore, abnormal APP processing could be involved in this progress. Periodontitis is a potentially transmissible chronic infection caused by plaque biofilm. It is responsible for 70% of the global population presenting one or more damages in the periodontium . ConsideGiven the accelerating trend of population aging, the harm caused by cognitive disorders on human health has become increasingly prominent. It clinically manifests as gradual cognitive dysfunction and psychosis . EpidemiPorphyromonas gingivalis , the most important periodontal pathogen, and its virulence factor lipopolysaccharide (LPS) are main pathogenic factor of periodontitis , P. gingivalis-LPS plus TAK-242 group (LPS+TAK-242), and TAK-242 group. TAK-242 , a novel cyclohexene derivative, is an effectively/specifically TLR4 inhibitor  into the palatal gingival sulcus of the maxillary first molars for 10 weeks using a 5-μl microsyringe . Moreover, we injected TAK-242  in rats in the LPS+TAK-242 group in advance. The rats in the TAK-242 group were injected with TAK-242 . The rats in the control group received an equivalent saline volume. The LPS and TAK-242 amounts were determined based on previous studies  and dissolved them according to the manufacturer’s instructions.We obtained 10-week-old male Sprague-Dawley rats of specific pathogen-free (SPF) grade from the Shanghai SIPPR-BK Laboratory Animal Co., Ltd. The rats were provided with standard housing at temperature (18–22°C) and humidity (55–65%) with a 12-h light/dark cycle and free access to food and water. We randomly divided 32 rats into four groups, namely, the control group (Control), nhibitor . After i studies . Three dThe open field in the present study consisted of a rectangular arena (530 mm × 478 mm), enclosed by a black wall, 590 mm in height . The test was initiated by gently placing a single rat in the middle of the arena, allowing the animal to move freely for 5 min while being recorded.The MWM test was conducted in a round pool 160 cm in diameter and 55 cm in depth . The pool was filled with water made opaque with white non-toxic water-based tempura paint. The water temperature was controlled to remain with a range equivalent to that of room temperature (22 ± 1°C). The platform was placed in the center of one quadrant of the pool and submerged 2.5 cm beneath the water surface; it remained in the same position throughout the learning trials and was removed from the pool during the probe test. A video-tracking system  was used to monitor and record the swimming activity of the rats. The rats should have learned to use the visual tips around the pool to find the hidden platform within 90 s, otherwise it would be gently guided to the platform and allowed to re-orient for an additional 10 s. Each rat was trained four times per day with a 30 s of rest per training interval. To examine spatial reference memory, a probe test was carried out on the sixth day when the platform was removed from the pool and each rat was placed into the water at the two quadrants furthest from the platform used on days 1–5, being allowed to navigate freely for 60 s.The maxillae of rats were obtained to detect bone parameters by micro-CT. Fixed in 4% paraformaldehyde, the bone morphometry was assessed using Skyscan1172  with an accuracy of 18 μm. Parameters including Bone volume/total volume (BV/TV), Bone surface/volume ratio (BS/BV) and Bone mineral density (BMD) were calculated.Peripheral blood from rats (5 ml per rat) was collected from abdominal aorta in the presence of heparin as the anticoagulant. Three milliliters of the whole blood was diluted with sterile PBS of the same volume and gently resuspended. Six milliliters of the diluted whole blood fraction was overlaid onto 3 ml of the Ficoll-Paque Plus  and then subjected to 800 × g for 20 min at RT with the centrifuge brake “off.” Then PBMC layers were washed twice with RPMI 1640 media by centrifugation at 1200 rpm for 5 min at 4°C. After isolation, all samples were dissolved in Trizol reagent  for lysis of cells to extract RNA.TM  on a Roche LightCycler 480 Real-Time PCR Detection System  according to the manufacturer’s protocol. Data were then processed using the 2–ΔΔCT method. All results were based on at least three independent tests, and the final results were expressed as normalized fold values relative to the control group. The sequences of genes including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), IL-1β, IL-6, IL-8 and IL-21, APP, amyloid precursor-like protein 1 (APLP1), APLP2, a disintegrin and metalloproteinase 10 (ADAM10), β-site APP cleaving enzyme 1 (BACE1), presenilin 1 (PS1), PS2, TLR4, cluster of differentiation 14 (CD14) and their primer pairs were listed in The RNA was extracted from PBMCs and homogenization of cortex using Trizol reagent  and the Total RNA Kit , respectively. The purity and concentration of RNA, as well as the cDNA synthesis, were conducted according to Of the approximately 5 ml of blood collected as previously described, 2 ml of blood were collected in heparinized tubes for the measurement of plasma cytokines. After centrifuging , supernatant medium was separated and immediately aliquoted into 1.5 ml cryogenic tubes and frozen at –80°C until use. For tissue, radioimmunoprecipitation assay (RIPA) lysis buffer , 1% protease inhibitor cocktail , and 1% PMSF  were used to homogenize samples of the cerebral cortex. Protein qualification was performed by BCA Protein Assay Kit . Equal amounts of protein were used in ELISA to measure levels of IL-1β, IL-6, IL-8, IL-21  and Aβ1-40, Aβ1-42  both in the cortex and plasma according to the manufacturer’s instructions. Level of LPS in the plasma of rats was detected by ELISA as well .The samples of the cerebral cortex of rats in four groups were homogenized and lysed by RIPA containing 1% protease inhibitor cocktail and 1% PMSF . Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis and transferred onto PVDF membrane blocked with 5% skimmed milk as previously described . A pre-s2O2 in methanol, blocked with 10% goat serum and incubated overnight at 4°C with the following primary antibodies: Ionized calciumbinding adaptor molecule 1 (Iba1) , Glial fibrillary acidic protein (GFAP)  to label microglia and astrocytes, anti-beta Amyloid1-42 antibody , anti-Tau antibody  and anti-APP antibody . After being washed, sections were incubated with biotinylated goat anti-rabbit or goat secondary antibody . Images were obtained with a Leica camera.Rats were anesthetized with 10% chloral hydrate and perfused with cool PBS before removal of the brain. One hemisphere was placed in 4% paraformaldehyde overnight at 4°C, after which paraffin sections were prepared. This procedure is consistent with the previous study . BrieflyThe quantification of the immunohistochemical analysis for Iba1, GFAP, Aβ1-42, APP and Tau positive cells was performed by Image J software. The endpoints and process length were evaluated, and the numbers of positive cells were determined according to previous studies . The aveP-values were calculated with one-way ANOVA and two-way ANOVA with the GraphPad Prism software. An analysis of variance was performed using Turkey’s post-hoc multiple comparison test. A value of p < 0.05 was indicative of statistical significance.All data are presented as the mean ± standard error of the mean (SEM). P. gingivalis-LPS led to posterior maxillary bone loss. There was a decrease in BV/TV and BMD  and an increase in BS/BV (14.92 ± 1.22/mm vs. 20.7 ± 0.40/mm) in the LPS group, which was reversed by TAK-242  (Periodontitis induced by d APLP2) . Furtherd APLP2) . Increasd APLP2) . Besidesd APLP2) . All thed APLP2) . Moreoved APLP2) . CompareResults of RT-PCR revealed that periodontal inflammation significantly increased the mRNA levels of TLR4 and CD14. Compared to the control group, the LPS group showed 2-fold and 1.5-fold increases in TLR4 and CD14 mRNA expression in the PBMCs and the cortex, respectively, which could be reversed by TAK-242 . As showP. gingivalis-LPS and learning and memory impairment in vivo. Micro-CT showed alveolar bone resorption. The MWM test showed that periodontitis impaired learning and memory ability. There was an increase in cortical and peripheral blood levels of inflammatory factors, as well as TLR4/NF-κB signaling pathway activation, which could be reversed by the TLR4 inhibitor TAK-242. Significant microglia and astrocyte activation were found in the cortex, which induced neuroinflammation. Furthermore, APP processing could be involved in this progress.In this study, we demonstrated the association between periodontitis induced by topical application of P. gingivalis-LPS into the gingival sulcus of maxillary first molars was used to cause periodontal inflammation according to a previous study (The association between systemic infection/inflammation and cognitive impairment has been reported . In the us study . The mosus study .P. gingivalis-LPST according to the results of the OFT. During the MWM test, however, the learning and memory ability could be impaired by periodontitis. Behavioral assessments demonstrated that topical application of P. gingivalis-LPS could induce learning and memory impairment. To investigate the association more comprehensively, more cognitive evaluation should be included in behavioral assessments using AD models in the following studies.The locomotor activity of rats were not affected by topical application of Periodontitis elicits a significant inflammatory response . It has Neuroinflammation could play a significant role in cognitive dysfunction . InflammP. gingivalis-LPS could stimulate brain secretion of inflammatory cytokines via TLR4 activation, which subsequently induces neuroinflammation.As one of the main triggers of inflammatory response, not only could the TLR4 signaling pathway regulate peripheral inflammation, but also neuroinflammation . SpecifiP. gingivalis -LPS could facilitate abnormal APP processing. This indicated that periodontitis induced by P. gingivalis-LPS could modulate APP processing through enhanced β- and γ-site secretase activity, consistent with the increase of APP in patients with chronic periodontitis (Besides, activated TLR4 signaling pathway and dysregulated cytokines might stimulate inflammatory processes by affecting APP processing . Multipldontitis . AccordiAbnormal APP processing could generate Aβ, which plays a pivotal role in the pathogenesis of cognitive disorders. It has traditionally been thought that Aβ in the brain originates from the brain tissue itself . HoweverIn this work, we established a periodontitis model and indicated the association between periodontitis and learning and memory impairment. Increase of inflammatory factors, activation of microglia and astrocytes and abnormal APP processing were found, suggesting that periodontitis might be a possible contributor to AD-like pathology. However, AD models should be used in following researches for further assessment of AD-pathologies and more sophisticated evaluation of association between periodontitis and AD.P. gingivalis-LPS could contribute to learning and memory impairment via neuroinflammation induced by TLR4/NF-κB signaling pathway activation in SD rats. Furthermore, abnormal APP processing could be involved in this progress. Therefore, periodontitis could not only affect the teeth-supporting structures but also cause a significant inflammatory load in both the peripheral blood and central nervous system, which leads to learning and memory impairment.Our findings indicated that periodontitis induced by topical application of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by the Animal Care and Welfare Committee of Shanghai Jiao Tong University School of Medicine.YH, WZ, and ZS wrote the main manuscript text. YH, HL, JZ, XZ, XX, and CQ performed the research. YH, HL, YL, and HC analyzed the data. WZ and ZS designed the main research, provided the necessary guidance on the performance of all the experiment, and contributed the essential reagents and tools. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.